Principal Supervisor: Dr Claudia Blindauer, Department of Chemistry
Co-supervisor: Dr Jose Gutierrez-Marcos, SLS
PhD project title: The role of plant metallothioneins in drought tolerance and zinc seed filling
University of Registration: University of Warwick
Minerals are essential for all organisms, including plants and animals. It is estimated that over 2 billion people suffer from micronutrient deficiency, with severe health and socio-economical consequences. Leading economists participating in the Copenhagen Consensus 2008 and 2012 concluded that fighting against micronutrient malnutrition is the top priority among the world’s most important current challenges.
In recent years, the importance of the micronutrient zinc has come to the fore. Zinc is a so-called “type 2” nutrient: it is involved in almost every metabolic process; hence, the effects of zinc deficiency are serious and multifactorial. In plants, zinc deficiency severely impairs growth; this is illustrated by increases in crop yields by up to 600% through fertilisation with zinc. In animals and man, zinc deficiency affects epidermal, gastrointestinal, immune, central nervous and reproductive systems. The WHO estimates that deficiency of this single trace element leads to the deaths of almost 0.5 million children every year.
Given that cereal seeds constitute a staple diet for the majority of people, it is therefore of interest to understand molecular mechanism of zinc accumulation and storage in cereal seeds.
The Blindauer group studies structure and dynamics of proteins involved in zinc homeostasis. Chief amongst these are plant metallothioneins (pMTs). These cysteine-rich proteins are major contributors to metal chelation in seeds, important in governing metal contents in developing grains and in metal re-mobilisation in germinating seeds. Moreover, they may also play roles in plant stress tolerance. Most importantly, there are indications that certain pMTs may contribute to drought tolerance, another critical aspect within food security.
The Gutierrez-Marcos group has extensive experience in gene targeting, mutagenesis and transformation in maize, one of the three most important staple seed cereals.
This project will investigate MTs from maize, and their potential roles in selective zinc seed filling and drought tolerance. We will take a highly cross-disciplinary approach ranging from in-planta analysis to detailed biophysical studies at the protein level.
- Establish maize MTs expression patterns in plant organs and in response to drought, oxidative stress, and treatment with phytohormones. This objective will help identify physiological functions of the respective MTs in maize.
- Characterise the metal-binding and structural properties of maize MT proteins. This will involve recombinant expression and purification of selected MTs, followed by NMR-spectroscopic and ESI-MS studies. Outcomes will provide understanding of molecular details of how these proteins exert their functions.
- Study the effects of manipulating expression of maize MTs in-planta to increase the selective accumulation of beneficial metal ions. MTs are capable of binding both essential (Zn, Cu) and toxic (Cd) metal ions. Total metal contents of seeds from wild-type and transgenic plants will be determined by elemental analysis, and the speciation of Zn will be studied using an innovative “metalloproteomics” approach. This will establish binding partners of essential Zn in seeds.
This objective will shed light on in-vivo metal-binding selectivity of maize MTs, their impact on plant health and vigour, and on seed Zn mobility and bio-availability.
- Determine the role of MTs in drought tolerance. We predict that increasing MTs in vegetative tissues will contribute to enhanced drought tolerance because they could (a) act as thiol-rich scavengers of reactive oxygen species normally induced by stress, and (b) supply zinc to re-start metabolism after desiccation. Selected MTs will be expressed in-planta to assess if they enhance drought tolerance in maize. The in-planta work will be complemented by biophysical studies to measure antioxidant activity and zinc-transfer ability of these MTs.
BBSRC Strategic Research Priority: Food Security
Techniques that will be undertaken during the project:
- Transcriptomics using RNAseq
- mRNA in situ hybridisation
- Generation of transgenic constructs and plant transformation
- Recombinant protein expression
- Protein purification by FPLC
- ICP-OES and -MS (Inductively-Coupled Plasma Optical Emission spectroscopy and - mass spectrometry for Elemental Analysis)
- Electrospray Ionisation Mass Spectrometry (ESI-MS) and MALDI-MS
- Metalloproteomics (combining chromatography, inorganic and biomolecular mass spectrometry) for zinc speciation analysis
- Protein NMR spectroscopy
- Other spectroscopies (UV-Vis, fluorescence, circular dichroism) for assays and protein analysis